Hematopoietic cell transplantation (HCT) is utilized to treat malignant and non-malignant diseases, but HCT is a high cost procedure with variable immune reconstitution outcomes that include morbidity and mortality risks. A better understanding of the bone marrow (BM)-derived hematopoietic stem cells (HSCs) and environment, termed niche, that affects immune reconstitution is warranted for improvement in therapeutic outcomes. Hematopoietic stem cells (HSCs) are multipotent cells that can differentiate into all hematopoietic lineages. In the BM niche, HSCs are in intimate contact with stromal mesenchymal stem cells (MSCs), which secrete CXCL12 to stimulate G protein-coupled receptor (GPCR) CXCR4 on HSCs. CXCL12/CXCR4 ligation activates signaling via intracellular G-proteins and plays a crucial role in the maintenance of quiescent HSCs and stem cell retention, homing, and mobilization. G protein-coupled receptor kinase 3 (GRK3) regulates CXCR4 by triggering desensitization, recruitment of -arrestin, and GPCR internalization; however, the regulatory function of GRK3 in stem cells, as well as other GPCRs critical to hematopoiesis, is unknown. We hypothesize regulation of GPCR signaling interactions by GRK3 controls cellular functions of both MSCs and HSCs. Our published work shows GRK3 deficient (-/-) mice have reduced CXCR4 internalization on hematopoietic cells, which consequently enhances CXCL12/CXCR4 signaling and cellular migration. We also observed GRK3-\- mice have a hypercellular BM with increased HSCs. My ex vivo preliminary data show GRK3-/- HSCs have increased proliferation in response to CXCL12, which is reduced to wildtype (WT)-level in the presence of CXCR4 antagonist. My preliminary short-term transplant investigations of GRK3-/- donor marrow transplanted into GRK3-/- recipients showed significantly increased hematopoietic cell proliferation compared to control WT donor marrow into WT recipients. Importantly, GRK3-/- donor marrow into WT recipients and WT donor marrow into GRK3-/- recipients had intermediate phenotypes of increased proliferation compared to control, suggesting an effect of GRK3 deficiency on HSCs and stromal BM niche components. GRK3-/- MSCs have enhanced proliferation and a propensity to develop into CXCL12-abundant reticular (CAR)-like cells and pre-osteoblasts, both known to enhance hematopoiesis. Although we have data to support a regulatory role of GRK3 on CXCL12/CXCR4 signaling interactions on leukocytes and HSCs, the GPCR(s) that GRK3 regulates on MSCs to induce the enhanced phenotypes previously discussed is unknown. To test our hypothesis, I will define the mechanism by which GRK3 regulation of CXCL12/CXCR4 signaling affects HSC function(s) (AIM 1) and determine the GPCR(s) on MSCs regulated by GRK3 (AIM 2). Lastly, I will investigate whether GRK3 deficiency improves HCT outcomes by performing long-term HCT studies (AIM 3). If we reveal GRK3 regulates two stem cell populations with crucial roles in hematopoiesis-- HSCs and MSCs-- then we will have identified a candidate therapeutic target for the potential enhancement of HCT.